Imaging method, device and system

ABSTRACT

The present disclosure discloses a method and a system for imaging. The method for imaging objects using the system for imaging. The system for imaging comprises a lens. The objects comprise a first object, a second object and a third object located at different positions on a first preset track. The method for imaging comprises: allowing the lens and the first preset track to move relatively in a first predetermined relationship to acquire a clear image of the third object using the system for imaging without focusing, the first predetermined relationship is determined by a focal plane position of the first object and a focal plane position of the second object. The aforementioned method for imaging is high in imaging efficiency and is capable of fast focusing according to the first predetermined relationship even if focus tracking fails so that the blurring of a photographed image due to defocusing is avoided.

TECHNICAL FIELD

The present disclosure relates to the field of optical detection, and inparticular to a method for imaging, a device for imaging, and a systemfor imaging.

BACKGROUND

In the related art, each time when photographing, a camera will quicklyadjust the focus to get the clearest focal plane, and thus a sharp/clearpicture can be acquired. This process is referred to as focus tracking.

However, when photographing with a camera in practical application,there might be some outside interferences. For example, whenphotographing, the camera fails to track the focus due to the existenceof scatter light around an object or dust or scratches on the surface ofthe object, and in this case, the image will be blurred if the cameracannot retrack the focus. For example, when a camera is used to performsequence determination, if an object is a nucleic acid molecule in aflowcell, it is easy for the camera to fail to track the focus when, forexample, there are bubbles and agglomerates of fluorescent impurities inthe liquid inside the photographed flowcell or dust and scratches on thesurface of the flowcell.

SUMMARY

Sequencing platforms for obtaining nucleic acid information based onimaging, such as currently commercially available second-generation orthird-generation sequencing platforms for obtaining nucleic acidinformation by photographing, comprise the process of photographing anucleic acid placed in a reactor using an system for imaging. Commonly,the reactor is also called a flowcell, which may comprise one or moreparallel channels for a reagent to get into and out of and for carryingthe reagent so as to form an environment needed by sequencedetermination reaction. The flowcell may be made by sticking two piecesof glass. The sequencing process comprises multiple rounds of cameraphotographing for a fixed region of the flowcell. The regionphotographed each time may be called a FOV (field of view), each roundof photographing may be called a cycle, and the reintroduction ofreagent is involved between two cycles to perform a chemical reaction.

In the normal photographing process, the camera can succeed, in mostcases, in automatically tracking the focus, i.e., finding the positionof the clearest focal plane. When there's interference, the camera mayfail to track the focus.

FIGS. 1-3 show data of successful focus tracking and abnormal focustracking or unsuccessful focus tracking in an experiment.

Taking continuously photographing two rows of FOVs in the same cycle asan example, the coordinates of the altitude (Z value) of an objectivewere recorded during photographing. As shown in FIG. 1 , the horizontalaxis is the serial numbers of FOVs, the first half FOVs are photographedin an order from the left side to right side of a flowcell and thesecond half FOVs are photographed from the right to the left in anotherrow. The vertical axis is the distance of the objective of a microscopefrom a camera, i.e., Z value (unit: um); the negative values indicatethat the objective of the microscope is located under the camera; andthe larger the absolute value of the Z value, the farther the objectiveis away from the camera.

FIG. 1 shows a Z value curve when 300 FOV images are successfullyphotographed with focus tracking. FIG. 2 shows a Z value curve when 200photographed FOV images comprise partial focus tracking abnormalities(represented as partial Z value abnormalities), and in this case, theimages which correspond to the abnormal portions of the curve shown asthe convex portions in the curve are unclear/blurred images.

Due to the certain limitation of camera focus tracking, defocusing couldeasily take place in the event of interference. After defocusing,because the objective was far away from a focal plane, that is, thedistance between the objective and the focal plane position was too farwhen photographing subsequent FOVs with focus tracking, the objectivecould not return to the focal plane even if the interference waseliminated. This case is shown as FIG. 3 . In FIG. 3 , the first 1 to200 FOVs belong to a cycle, and the rest of the FOVs belong to anothercycle. As shown in FIG. 3 , after the 268th FOV (located in the firstrow in another cycle), focus tracking failed, and after interferencedisappeared, focus retracking still failed before the end of the cycle.

Focus tracking failure means a blurred image, which will causeinformation loss. Therefore, this is a problem which must be solved. Inreality, although interference cannot be completely eliminated,generally it is hoped that at least a clear image can still be acquiredafter interference disappears.

By analyzing a lot of data involving successful focus tracking andabnormal focus tracking, it has been discovered that when the objectiveis fixed, a plurality of Z value curves corresponding to a plurality ofFOVs being photographed with same normal focus tracking in differentcycles (i.e., different times) show a certain law. FIG. 4 shows Z valuecurves corresponding to clear images acquired by photographing 300 FOVswith normal focus tracking in four different cycles.

Two laws are discovered:

(1) For the same position (FOV), there might be different focal planesin different cycles; however, relative to other FOVs in the same cycle,the relative position of its focal plane is basically unchanged. Thatis, in terms of physical positions, the focal planes of different FOVsin the same cycle are correlated.

(2) In the drawing, half of the 300 FOVs of each curve are photographedfrom the left side to right side of a row of the flowcell, while theother half are photographed from the right side to the left side inanother row; and due to the deformation of the flowcell and/or thealtitude difference between the left side and the right side, the focalplanes of a plurality of continuous FOVs in the same direction of thesame row (e.g., from the left to the right or from the right to theleft) show a certain law, which can fit a straight line well.

It is guessed that the aforementioned laws result from the followingpossible causes: because the same FOV needs to be repeatedlyphotographed in different cycles, after heating and introduction ofreagent, the internal pressure of the flowcell change, and the wholefocal plane shift. Relative to the whole flowcell, each FOV is verysmall, so the surface evenness of each FOV can be regarded to beunchanged, which means that the relative focal plane positions ofadjacent FOVs are unchanged.

Based on the aforementioned discovered laws, a set of algorithm isdeveloped, which enable a camera to have a focal plane predictionfunction with the aid of a software algorithm without replacinghardware. Specifically, for example, in cycle 1, for a plurality of FOVslocated on the same preset track (a first preset track, e.g., the samerow), focal planes of two of the FOVs can be obtained with focusing, thefocal plane difference between the focal planes is calculated, arelationship (e.g., a first predetermined relationship) is obtained bylinear fitting, and the focal plane positions of the other FOVs in therow are predicted using the relationship. For cycle 2 and subsequentcycles, by memorizing the normally focused focal plane of any FOV in theaforementioned cycle 1 or any of previous cycles and then focusing todetermine the focal plane position of the FOV in the current cycle,relationships can be established by linear regression to predict thefocal plane of any other FOV in the current cycle.

The establishment of a relationship by linear regression is expressed asformula (a): y=kx+b, and slope k (also referred to as change tendency k)and an intercept b (also referred to as basic offset b) need to bedetermined. Based on the aforementioned law (1), it can be known that kis equal to 1, so formula (a) can be converted into formula (b): y=x+b,and b can be determined based on the relative positions and Z values offocal planes of any two FOVs on the same track in the same cycle.

For example, for the cycle 1, the basic offset b may be calculated by anoverall focal plane difference (e.g., from one end of the track to theother end of the track). Specifically, cyc1FovZ(r) and cyc1FovZ(1),which respectively represent Z values of the focal plane positions oftwo objects (referred to as two positions or two FOVs) at one end andthe other end of a track in cycle 1, are obtained with focusing, so thatan intercept can be calculated according tob=(cyc1FovZ(r)—cyc1FovZ(1))/FovNum, wherein FovNum denotes the number ofFOVs between the two positions of cyc1FovZ(r) and cyc1FovZ(1). Formula(b) can be used to predict cyc1FovZ(n+1) of the cycle 1, cyc1FovZ(n) andcyc1FovZ(n+1) in (b) denote two adjacent positions (FOVs) andcyc1FovZ(n+1) is relatively closer to cyc1FovZ(r), and cyc1FovZ(n) canbe obtained with focusing.

It should be noted that b can be determined by the focal planeinformation of two FOVs on the same track. Moreover, determined formula(b) and the focal plane coordinate information of any focused FOV canalso be used. Here, for example, the determined relationship (b) and anyvalue among determined cyc1FovZ(r) and cyc1FovZ(1) are used to determinecyc1FovZ(n+1).

After the linear relationship of one cycle is determined, for thesubsequent photographing of any cycle of the same track/the same FOVs,the focal plane position of any FOV in the current cycle can bepredicted based on the determined linear relationship and the focalplane position of any one FOV in the current cycle. For example, thefocal plane position of Fov(n+1) (the (N+1)th FOV or the FOV at the(N+1)th position) in the current cycle is predicted using the focalplane position of Fov(n) (the Nth FOV or the FOV at the Nth position) inthe same cycle, and the Z value curFovZ(n) of the Nth FOV can besubstituted as a dependent variable into formula (b), and the obtained yis curFovZ(n+1).

In addition, after the linear relationship of one cycle is determined,for the subsequent photographing of any cycle of the same track/the sameFOVs, the focal plane positions of two FOVs in the cycle and the focalplane position of one of the same FOVs in the current cycle can also bedetermined based on the determined linear relationship to predict thefocal plane position of the other of the same FOVs in the current cycle.For example, formula (b) is determined in the previous cycle; the focalplane position of Fov(n+1) (the (N+1)th FOV or the FOV at the (N+1)thposition) in the current cycle is predicted using the focal planeposition of Fov(n) (the Nth FOV or the FOV at the Nth position) in thesame cycle; the focal plane positions of Fov(n) and Fov(n+1) in theprevious cycle, which are respectively represented by preFovZ(n) andpreFovZ(n+1), can be determined by formula (b), and curFovZ(n+1) isdetermined using formula (c):curFovZ(n+1)=curFovZ(n)+(preFovZ(n+1)−preFovZ(n)).

It should be noted that the discovery and explanation of theaforementioned laws and linear relationships as schematicallyestablished relationships are merely intended to facilitate descriptionor understanding. It can be understood by those skilled in the art thatthe first preset track may be a straight line or a curve, and any curvemay be regarded as the fitting of a plurality of segments. In thisregard, it is believed that, through the aforementioned illustration ofthe related scenarios (including the discovery of the laws and theestablishment of the relationships) of the present disclosure, for thecase that the first preset track is a curve, those skilled in the artcan follow the idea of the present disclosure to regard the curve-shapedfirst preset track as a group of segments and correspondingly establisha first preset relationship including a group of linear relationships soas to predict the focal plane positions of objects on the track withoutfocusing.

Without replacing hardware, the camera can return to the vicinity of afocal plane by the method for imaging of the present embodiment and thenstart photographing. Based on the discovery and the explanation above,the present disclosure provides a method for imaging, an imaging device,a system for imaging, and a sequencing system.

The method for imaging according to an embodiment of the presentdisclosure images an object using the system for imaging. The system forimaging comprises a lens. The object comprises a first object, a secondobject and a third object located at different positions on a firstpreset track. The method for imaging comprises: allowing the lens andthe first preset track to move relatively in a first predeterminedrelationship to acquire a clear image of the third object using thesystem for imaging without focusing, wherein the first predeterminedrelationship is determined by a focal plane position of the first objectand a focal plane position of the second object.

The system for imaging according to an embodiment of the presentdisclosure images an object. The system for imaging comprises a lens anda control device. The object comprises a first object, a second objectand a third object located at different positions on a first presettrack. The control device is used to: allow the lens and the firstpreset track to move relatively in a first predetermined relationship toacquire a clear image of the third object using the system for imagingwithout focusing, wherein the first predetermined relationship isdetermined by a focal plane position of the first object and a focalplane position of the second object.

In the aforementioned method for imaging and system for imaging, thefirst predetermined relationship is determined by the focusing positionsof the first object and the second object, and when other objects on thefirst preset track are imaged, focal planes can be directly predictedaccording to the first predetermined relationship to acquire a clearimage of the third object without focusing. The method for imaging isparticularly suitable for a scenario where there are a lot of objectsand it is desired to quickly and continuously acquire images of theseobjects. The method is high in imaging efficiency and is able toaccurately determine the focal plane positions of the subsequent objectsto acquire the image information of the subsequent objects in continuousimage acquisition even if the system for imaging fails to track thefocus. The method used in cooperation with a focus tracking system ofthe system for imaging can provide a remedy in the event that the focustracking system cannot normally track the focus again after it fails totrack the focus.

The sequencing device according to an embodiment of the presentdisclosure comprises the system for imaging of the aforementionedembodiment.

The computer-readable storage medium according to an embodiment of thepresent disclosure is configured for storing a program for execution bya computer, and the execution of the program comprises implementing thesteps of the method according to the aforementioned embodiment. Thecomputer-readable storage medium may include: read-only memory, randomaccess memory, magnetic disk, optical disk, or the like.

The system for imaging according to an embodiment of the presentdisclosure is configured for imaging objects, wherein the system forimaging comprises a lens and a control device, the objects comprise afirst object, a second object and a third object located at differentpositions on a first preset track, the control device comprisescomputer-executable programs, and the execution of thecomputer-executable programs comprises implementing the steps of themethod according to the aforementioned embodiment.

The computer program product according to an embodiment of the presentdisclosure comprises instructions, which enable a computer to implementthe steps of the method according to the aforementioned embodiment whenexecuted by the computer.

The additional aspects and advantages of the embodiments of the presentdisclosure will be partially set forth in the following description, andwill partially become apparent from the following description or beappreciated by practice of the embodiments of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The aforementioned and/or additional aspects and advantages ofembodiments of the present disclosure will become apparent and easilyunderstood from the description of the embodiments in reference to thefollowing drawings, among which:

FIG. 1 is a Z value graph when focus tracking succeeds during sequencedetermination;

FIG. 2 is a Z value graph when focus tracking fails in abnormal convexportions FOV during sequence determination;

FIG. 3 is a Z value graph when focus retracking still fails at the endof cyclic photographing after focus tracking fails and interferencedisappears during sequence determination;

FIG. 4 is a schematic graph of different focusing positions generatedfrom focusing data of objects during sequence determination;

FIG. 5 is a structural schematic diagram of a first preset track and asecond preset track according to one embodiment of the presentdisclosure;

FIG. 6 is a schematic graph of focusing positions generated fromfocusing data of the objects in the absence of interference duringsequence determination;

FIG. 7 is a schematic graph of focusing positions generated fromfocusing data of the objects when focus retracking succeeds in thepresence of interference during sequence determination;

FIG. 8 is a schematic graph of focusing positions generated fromfocusing data of the objects when focus retracking fails in the presenceof interference during sequence determination;

FIG. 9 is a schematic flow chart of a focusing method according to oneembodiment of the present disclosure;

FIG. 10 is a schematic diagram of a positional relationship between alens and an object according to one embodiment of the presentdisclosure;

FIG. 11 is a partial structural schematic diagram of a system forimaging according to one embodiment of the present disclosure;

FIG. 12 is a schematic diagram of a connected component of an imageaccording to one embodiment of the present disclosure;

FIG. 13 is another schematic flow chart of the focusing method accordingto one embodiment of the present disclosure;

FIG. 14 is yet another schematic flow chart of the focusing methodaccording to one embodiment of the present disclosure;

FIG. 15 is still another schematic flow chart of the focusing methodaccording to one embodiment of the present disclosure; and

FIG. 16 is still yet another schematic flow chart of the focusing methodaccording to one embodiment of the present disclosure.

DETAILED DESCRIPTION

The embodiments of the present disclosure are described in detail below,and the examples of the embodiments are shown in the accompanyingdrawings, throughout which identical or similar reference numeralsrepresent identical or similar elements or elements having identical orsimilar functions. The embodiments described below by reference to theaccompanying drawings are exemplary and are merely intended to explainthe present disclosure rather than be construed as limiting the presentdisclosure.

This application claims the benefit of priority from Chinese PatentApplication No. 201810814359.0 and 201810813660.X filed on Jul. 23,2018, the entire contents of which applications are hereby incorporatedby reference in this application.

In the description of the present disclosure, it should be understoodthat the terms “first”, “second”, “third”, “fourth” and “fifth” aremerely intended to facilitate description rather than be construed asindicating or implying relative importance or implicitly indicating thenumber of indicated technical features. Therefore, features defined with“first” and “second” may explicitly or implicitly include one or more ofthe features. In the description of the present disclosure, unlessotherwise specifically defined, “a plurality of” means two or more thantwo.

In the description of the present disclosure, unless otherwise clearlyspecified and defined, “connect” should be comprehended in its broadsense. For example, “connect” may be “fixedly connect”, “detachablyconnect” or “integrally connect”; “mechanically connect”, “electricallyconnect” or “communicate with each other”; or “directly interconnect”,“indirectly interconnect through an intermediate”, “the communicationbetween the interiors of two elements” or “the interaction between twoelements”. For those of ordinary skill in the art, the specific meaningsof the aforementioned terms in the present disclosure can be understoodaccording to specific conditions.

Directions or positional relationships indicated by terms such as“center”, “thickness”, “upper”, “lower”, “front”, “rear”, etc. are thoseshown based on the detailed description or the accompanying drawings,and are merely intended to facilitate and simplify description ratherthan indicate or imply that the indicated device or element must have aspecific direction and be structured and operated according to thespecific direction.

The “unchanged” involving, for example, distance, object distance and/orrelative position may be expressed as “absolutely unchanged” or“relatively unchanged” in numerical value, numerical value range orquantity, and the “relatively unchanged” is “kept within a certaindeviation range or a preset acceptable range”. Unless otherwise stated,“unchanged” involving distance, object distance and/or relative positionis “relatively unchanged”.

The disclosure hereinafter provides a plurality of embodiments orexamples for implementing the technical solutions of the presentdisclosure. The present disclosure may repeat reference numbers and/orreference letters in different examples. Such repetition is intended forsimplicity and clarity rather than for indicating the relationshipbetween various embodiments and/or settings discussed.

“Sequence determination” used in the embodiments of the presentdisclosure refers to nucleic acid sequence determination, including DNAsequencing and/or RNA sequencing, and/or including long fragmentsequencing and/or short fragment sequencing. The “sequence determinationreaction” refers to sequencing reaction.

One embodiment of the present disclosure provides a method for imaging,which images objects using a system for imaging. Referring to FIGS. 5,11 and 12 , the system for imaging comprises a lens 104. The objectscomprise a first object 42, a second object 44 and a third object 46located at different positions on a first preset track 43. The methodfor imaging comprises: allowing the lens 104 and the first preset track43 to move relatively in a first predetermined relationship to acquirean image of the third object 46 using the system for imaging withoutfocusing, wherein the first predetermined relationship is determined bya focal plane position of the first object 42 and a focal plane positionof the second object 44.

In the aforementioned method for imaging, the first predeterminedrelationship is determined by the focusing positions of the first object42 and the second object 44, and when other objects on the first presettrack are imaged, focal planes can be directly predicted according tothe first predetermined relationship to acquire a clear image of thethird object without focusing. The method for imaging is particularlysuitable for a scenario where there are a lot of objects and it isdesired to quickly and continuously acquire images of these objects. Themethod is high in imaging efficiency and is able to accurately determinethe focal plane positions of the subsequent objects to acquire the imageinformation of the subsequent objects in continuous image acquisitioneven if the system for imaging fails to track the focus. The method usedin cooperation with a focus tracking system of the system for imagingcan provide a remedy in the event that the focus tracking system cannotnormally track the focus again after it fails to track the focus.

Specifically, in the example of FIG. 5 , the first preset track 43 maybe a linear track, and the first object 42 and the second object 44 arelocated at two positions of the linear track, for example, at two endsof the linear track. It can be understood that there may be a pluralityof third objects 46, and the plurality of third objects 46 aresequentially arranged on the first preset track 43, and are locatedbetween the first object 42 and the second object 44. It can beunderstood that, in other examples, the third objects 46 may be locatedat other positions different from the positions of the first object 42and the second object 44. In other examples, the first preset track 43may be a non-linear track, e.g., a curve-shaped track, which can beregarded as the fitting of a plurality of segments, and the firstobject, the second object and the third object are located on a samesegment in the curve-shaped track.

In some embodiments, the first predetermined relationship may be alinear relationship. In one embodiment, referring to FIG. 5 , when thefirst preset track 43 is one or more channels 52 of a flowcell 500 usedin the process of sequence determination and the imaged third object 46is one or more positions (FOVs) in the channels 52, duringphotographing, the lens and the first preset track 43 can moverelatively in a first direction A. For example, the lens 104 is fixedand the lens 104 comprises an optical axis OP, and the first presettrack 43 moves in a direction perpendicular to the optical axis OP. Itcan be understood that in some embodiments, the first preset track 43can move in a direction parallel to the optical axis OP. The firstpreset track 43 can be moved according to the requirement of actualadjustment.

The system for imaging comprises a camera 108, the lens 104 may bemounted on the camera 108, and the camera 108 performs imaging byacquiring light passing through the lens 104.

In some embodiments, allowing the lens 104 and the first preset track 43to move relatively comprises at least one of the following: fixing thelens 104 and moving the first preset track 43, fixing the first presettrack 43 and moving the lens 104, and simultaneously moving the lens 104and the first preset track 43.

Thus, as the lens 104 and the first preset track 43 can be moved in avariety of ways, the adaptability is high, and the application range ofthe method for imaging is broadened.

Specifically, when the first preset track 43 needs to be moved, thefirst preset track 43 can be placed on a carrier and the carrier canbring the first preset track 43 and the objects to translate back andforth in a direction perpendicular to the optical axis OP of the lens104, so that one of the third objects 46 can be placed under the lens104, and thereby the system for imaging can image the third object 46.

When the lens 104 needs to be moved, the lens 104 can be mounted on adriving mechanism and the driving mechanism can electrically or manuallydrive the lens 104 to translate back and forth in a directionperpendicular to the optical axis OP of the lens 104, so that the lens104 can be over one of the third objects 46, and thereby the system forimaging can image the object.

Simultaneously moving the lens 104 and the first preset track 43 can beunderstood as first moving the lens 104 and then moving the first presettrack 43 to make one of the third objects 46 be located under the lens104; or first moving the first preset track 43 and then moving the lens104 to make the lens 104 be located over one of the third objects 46; ormoving the lens 104 and the first preset track 43 together to make thelens be located over one of the third objects 46.

In some embodiments, the determination of the first predeterminedrelationship comprises: focusing the first object 42 using the systemfor imaging to determine first coordinates; focusing the second object44 using the system for imaging to determine second coordinates; andestablishing the first predetermined relationship according to the firstcoordinates and the second coordinates, wherein the first coordinatesrepresent the focal plane position of the first object 42, and thesecond coordinates represent the focal plane position of the secondobject 44. Thus, the first predetermined relationship can be determinedin advance, and when imaging other objects, clear images of the otherobjects can be acquired using the system for imaging without focusingaccording to the first predetermined relationship, thus simplifying themethod for imaging and increasing the efficiency of the method forimaging.

Specifically, according to one embodiment mentioned above, referring toFIG. 5 , when the imaged third object 46 is one or more positions of theflowcell 500 used in sequence determination, the first object 42, thesecond object 44 and the third object 46 can be located in the samechannel of the flowcell 500.

Preferably, the first object 42, the third object 46 and the secondobject 44 are sequentially arranged on the first preset track 43.According to one embodiment mentioned above, the first direction A is adirection from the left to the right of the flowcell 500, that is, thefirst object 42, the third object 46 and the second object 44 aresequentially arranged on the first preset track 43 in the direction Afrom the left to the right of the flowcell 500. In other embodiments,the first object 42, the third object 46 and the second object 44 mayalso be arranged on the first preset track 43 in other orders. It can beunderstood that during the determination of the first predeterminedrelationship, two objects, the first object 42 and the second object 44,can be selected on the first preset track 43 for focusing so as toacquire the focusing positions of the two objects. Specifically, it canbe known from the foregoing description that during sequencedetermination, the relative focal plane positions of two FOVs(particularly adjacent FOVs) on the first preset track 43 are keptunchanged. Therefore, the first predetermined relationship can bedetermined with the focal plane coordinate data of the first object 42and the second object 44 acquired by focusing the first object 42 andthe second object 44. By using the first predetermined relationship, anythird objects on the first preset track 43 can be acquired withoutfocusing.

Therefore, as an example, the first object 42 and the second object 44may be a starting point FOV and an end point FOV on the first presettrack (e.g., FOVs at both ends of the same row of the same channel) in acycle (i.e., the same period of time), as shown in FIG. 5 . The thirdobject 46 may be any one or multiple FOVs between the first object 42and the second object 44. It can be understood that based on the abovelaw, the first object 42 and the second object 44 may also be FOVs atother positions, and the third object 46 does not need to be locatedbetween the first object 42 and the second object 44 either. It is onlynecessary to, based on a rule of determining a straight line (firstpredetermined relationship) according to two points, select any twopositions (objects) on the first preset track, acquire a focal planeposition corresponding to each position and acquire the firstpredetermined relationship corresponding to the first preset track 43according to the focal plane positions at each position, and thereby animage of the third object can be acquired using the system for imagingthrough the first predetermined relationship without focusing. In apractical application scenario, a coordinate system can be establishedto digitize/quantify relative positional relations including the focalplane positions. For example, when image signals are acquired using asequence determination platform, a three-dimensional coordinate systemcan be established with x and y representing the planes where the firstpreset track and a second preset track locate and z representing thedirection of the optical axis of an objective, and the focal planeposition at each position comprises a Z value for a focal plane.

It should be noted that the mentioned cycle represents the influence oftime factor/image acquisition cycle. Generally, in a high-precisionsystem for imaging, such as a microscopic system with a 60× objectiveand a depth of field of 200 nm, the fluctuation caused by one or moreback-and-forth mechanical movements of the first/second preset track orone or more back-and-forth mechanical movements of a platform bearingthe first/second preset track may exceed the depth of field. Therefore,preferably, when using the method for imaging according to any of theaforementioned or undermentioned embodiments to continuously image aplurality of objects multiple times at high precision, if the pluralityof objects located on the same preset track are not in the same imageacquisition time cycle (e.g., in different mechanical movementdirections), a first predetermined relationship that is established byre-fitting based on focusing data after refocusing is relatively moreaccurate and better. It can be understood by those skilled in the artthat when continuously imaging a plurality of objects at relatively lowprecision, due to a large depth of field, the focal plane positiondeviation caused by mechanical reciprocation may not be considered. Thatis, for the plurality of objects on the same preset track, the firstand/or second predetermined relationships determined in any previousimage acquisition cycle can be used for imaging in different imageacquisition cycles.

Predicted results of Z value after the aforementioned predictionstrategy is employed are shown as FIGS. 7-9 .

The C5 curves in FIGS. 6-8 are Z value curves (focal plane lines formedby actual focusing positions) acquired from real photographing of thecamera in which only camera focus tracking is used. The C6 curves arepredicted Z value curves (focal plane lines formed by predicted focusingpositions).

FIG. 6 shows the prediction of Z values of a plurality of FOVs in acycle in the absence of interference, and FIG. 7 and FIG. 8 show theprediction of Z values in the presence of interference and defocusing.Without intervention, focus retracking succeeds after defocusing in FIG.7 , while focus retracking fails after defocusing in FIG. 8 .

In some embodiments, the objects comprise a fourth object 47 and a fifthobject 48 located at different positions on the second preset track 45.The method for imaging comprises: allowing the lens 104 and the secondpreset track 45 to move relatively in a second predeterminedrelationship to acquire an image of the fifth object 48 using the systemfor imaging without focusing, wherein the second predeterminedrelationship is determined by a focal plane position of the fourthobject 47 and the first predetermined relationship, and the secondpreset track 45 is different from the first preset track 43. Based onthe first predetermined relationship of the first preset track 43 andthe focal plane position of any object on the second preset track 45,the second predetermined relationship corresponding to the second presettrack 45 is determined. A clear image of any object on the second presettrack 45 can be acquired using the second predetermined relationshipwithout focusing, and thus, clear images of more objects can beacquired, satisfying the demand of users.

Specifically, the second preset track 45 may be a track adjacent to thefirst preset track 43. In the aforementioned embodiment, the secondpreset track 45 is a parallel channel adjacent to the first preset track43. The second preset track 45 may be a linear track, and the fourthobject 47 and the fifth object 48 are located at two positions of thelinear track. For example, the fourth object 47 is located at one end ofthe linear track, and the fifth object 48 is located in the middle ofthe linear track. It can be understood that there may be a plurality offifth objects 48, and the plurality of fifth objects 48 are sequentiallyarranged on the second preset track 45 and the fifth objects 48 arelocated at positions different from that of the fourth object 47. It canbe understood that in other examples, the second preset track 45 may bea non-linear track, e.g., a curve-shaped track, which can be regarded asthe fitting of a plurality of segments, and the fourth object 47 and thefifth object 48 are located on a same segment in the curve-shaped track.

In some embodiments, the second predetermined relationship may be alinear relationship.

In one embodiment, referring to FIG. 5 , when the second preset track 45is one or more channels 52 of the flowcell 500 used in the process ofsequence determination and the imaged fifth object 48 is one or morepositions (FOVs) in the channels 52, during photographing, the lens andthe second preset track can move relatively in a second direction B. Forexample, the lens is fixed and comprises an optical axis, and the secondpreset track 45 can move in a direction perpendicular to the opticalaxis. It can be understood that in some embodiments, the second presettrack 45 can move in a direction parallel to the optical axis OP. Thesecond preset track 45 can be moved according to the requirement ofactual adjustment.

It can be understood that for other ways of allowing the lens 104 andthe second preset track 45 to move relatively, the aforementionedexplanation of the ways of allowing the lens 104 and the first presettrack 43 to move relatively can also be referred to. Therefore, therewill be no detailed description herein to avoid redundancy. It should benoted that in the example of FIG. 5 , the first preset track 43 and thesecond preset track 45 are two adjacent channels 52 on the flowcell 500,so the first preset track 43 and the second preset track 45 can movesynchronously when the flowcell 500 is moved.

In some embodiments, the determination of the second predeterminedrelationship comprises: focusing the fourth object 47 using the systemfor imaging to determine fourth coordinates; and establishing the secondpredetermined relationship according to the first predeterminedrelationship and the fourth coordinates, wherein the fourth coordinatesrepresent the focal plane position of the fourth object 47. Thus, thesecond predetermined relationship can be determined in advance, and whenimaging other objects, clear images of the other objects can be acquiredusing the system for imaging without focusing according to the secondpredetermined relationship, thus simplifying the method for imaging andincreasing the efficiency of the method for imaging.

Specifically, according to one embodiment mentioned above, referring toFIG. 5 , when the imaged fifth object 48 is one or more positions of theflowcell 500 used in sequence determination, the fourth object 47 and aplurality of fifth objects 48 can be located in the same channel of theflowcell 500.

Preferably, the fourth object 47 and the plurality of fifth objects 48are sequentially arranged on the second preset track 45. According toone embodiment mentioned above, the second direction B is a directionfrom the right to the left of the flowcell 500, that is, the fourthobject 47 and the plurality of fifth objects 48 are sequentiallyarranged on the second preset track 45 in the direction B from the rightto the left of the flowcell 500. In other embodiments, the fourth object47 and the fifth objects 48 may also be arranged on the second presettrack 45 in other orders.

It can be understood that for the determination of the secondpredetermined relationship, the aforementioned explanation of thedetermination of the first predetermined relationship can be referredto. Therefore, there will be no detailed description herein to avoidredundancy.

In some embodiments, the method for imaging comprises: allowing, afteracquiring the image of the third object 46, the lens 104 and the firstpreset track 43 and/or the second preset track 45 to move relatively toacquire an image of the fifth object 48 using the system for imagingwithout focusing. Thus, after the image of the third object 46 on thefirst preset track 43 is acquired, the image of the fifth object 48 onthe second preset track 45 can be acquired, and thereby, the imaging ofobjects on different preset tracks can be realized.

Specifically, in the aforementioned embodiment, after the images of oneor more third objects 46 on the first preset track 43 are acquired, thelens 104 and the flowcell 500 are moved relatively in a third directionC (i.e., a direction perpendicular to the extending direction of thechannel 52), so that the lens 104 is located over the fifth object 48,and the image of the fifth object 48 is then acquired using the systemfor imaging without focusing according to the second predeterminedrelationship. In the illustrated embodiment, the third direction C isperpendicular to the first direction A and the second direction B.

Further, in the example shown in FIG. 5 , the first preset tracks 43 andthe second preset tracks 45 are alternately arranged at intervals fromtop to bottom. After the images of one or more third objects 46 on theuppermost first preset track 43 are acquired, the lens 104 and theflowcell 500 are moved relatively, so that the lens 104 is located overthe fifth objects 48 on the first second preset track 45, and images ofone or more fifth objects 48 on the first second preset track 45 arethen acquired. Then the lens 104 and the flowcell 500 are movedrelatively, so that the lens 104 is located over the third object 46 onthe second first preset track 43 and an image of the third object 46 onthe second first preset track 43 is then acquired, until clear images ofthe objects on all the first preset tracks 43 and the second presettracks 45 are acquired.

To sum up, since the focal plane position (e.g., Z value) of the objectto be imaged is predicted according to the first predeterminedrelationship or the second predetermined relationship, other FOVs can beimaged without focusing, which increases the imaging efficiency andaccuracy. Further, when applied in the process of photographing the sameregion and similar regions, this determination method can quickly andcontinuously acquire images of a plurality of objects. The focusingprocess can also be omitted in the process of continuous photographingso as to realize fast scanning photographing. Further, in cooperationwith the automatic focus tracking system of the camera, the focal planeprediction technology adopted can achieve better image quality, and cansolve the problem that the camera cannot retrack the focus in the eventof interference and defocusing. In a broader sense, the camera can havecertain intelligence when using the focal plane prediction technology,which enables quick focusing and even the omission of focusing byassisting the focusing according to prior knowledge. Particularly in theprocess of photographing, such intelligence has more important extendedapplication.

In some embodiments, the system for imaging comprises a device forimaging 102 and a carrier. The device for imaging 102 comprises a lens104 and a focusing module 106. The lens 104 comprises an optical axisOP, and the lens 104 can move along the direction of the optical axisOP. A first preset track 43 and/or a second preset track 45 are locatedon the carrier.

The focusing process for the determination of a first predeterminedrelationship or a second predetermined relationship in the presentdisclosure will be illustrated with specific embodiments below. Itshould be noted that, unless otherwise specified, the elements with thesame name used in different embodiments should be limited to theexplanation of the respective embodiments, and the elements with thesame name in different embodiments should not be cross-understood orconfused.

Embodiment 1

Referring to FIGS. 9-11 , focusing comprises: (a) emitting light onto anobject using a focusing module; (b) moving a lens to a first setposition; (c) moving the lens from the first set position toward theobject at a first set step length, and determining whether the focusingmodule receives the light reflected by the object; (d) when the focusingmodule receives the light reflected by the object, moving the lens fromthe current position to a second set position, wherein the second setposition is located within a first range which is a range including thefirst set position and allowing the lens to move in the direction of anoptical axis; (e) moving the lens from the second set position at asecond set step length, and acquiring an image of the object at aposition of each step using a device for imaging, wherein the second setstep length is less than the first set step length; and (f) evaluatingthe images of the object to obtain an image evaluation result andfocusing according to the image evaluation result.

By using the aforementioned method for imaging, a plane for clearimaging of a target object, i.e., a clear plane/clear surface, can bequickly and accurately found. The method is particularly suitable for adevice comprising a precision optical system and having difficulty infinding a clear plane, such as an optical detection device with ahigh-magnification lens. Thus, cost can be reduced.

Specifically, in the aforementioned focusing step, the object is anobject whose focal plane position needs to be acquired. For example, ifa first predetermined relationship needs to be determined, two objectscan be selected on a first preset track, and focusing is successively orsimultaneously performed on the two objects on the first preset track 43to acquire two sets of focal plane position data, wherein one set offocal plane position data serves as the focal plane position data of thefirst object 42 and the other serves as the focal plane position data ofthe second object 44; and if a second predetermined relationship needsto be determined, an object can be selected on a second preset track forfocusing to acquire the focal plane position data of the object, whichserves as the focal plane position data of a fourth object 47, so thatthe second predetermined relationship can be determined with referenceto the first predetermined relationship.

Referring to FIG. 10 and FIG. 11 , in the embodiment of the presentdisclosure, objects are a plurality of positions (Field of Views, FOVs)of a sample 300 applied in sequence determination. Specifically, whenthe first predetermined relationship needs to be determined, the objectfor focusing may serve as a first object or a second object; and whenthe second predetermined relationship needs to be determined, the objectfor focusing may serve as a fourth object or a fifth object. The sample300 comprises a carrying device 200 and a test sample 302 located on thecarrying device. The test sample 302 is a biomolecule, such as a nucleicacid. The lens 104 is over the carrying device 200. The carrying device200 is provided with a front panel 202 and a back panel (lower panel),each of which is provided with two surfaces, and the test sample 302 isconnected to the upper surface of the lower panel, that is, the testsample 302 is located under the lower surface 204 of the front panel202. In an embodiment of the present disclosure, because the device forimaging 102 acquires an image of the test sample 302, the test sample302 is the corresponding position (FOV) during photographing. The testsample 302 is located under the lower surface 204 of the front panel 202of the carrying device 200, and when a focusing process begins, the lens104 moves in order to find a medium interface 204 where the test sample302 is located so as to increase success rate of the device for imaging102 in acquiring a clear image. In an embodiment of the presentdisclosure, the test sample 302 is a solution, the front panel 202 ofthe carrying device 200 is glass, and the medium interface 204 betweenthe carrying device 200 and the test sample 302 is the lower surface 204of the front panel 202 of the carrying device 200, i.e., the interfacebetween two media (glass and liquid). The test sample 302 from which thedevice for imaging 102 needs to acquire an image is located under thelower surface 204 of the front panel 202, and at this time, a clearsurface for the clear imaging of the test sample 302 is determined andfound by the image acquired by the device for imaging 102. This processcan be referred to as focusing. In one example, the thickness of thefront panel 202 is 0.175 mm.

In other embodiments, the carrying device 200 may be a slide, and thetest sample 302 is placed on the slide or the test sample 302 is clampedbetween two slides. In another embodiment, the carrying device 200 maybe a reaction device, e.g., a flowcell with an upper carrying panel anda lower carrying panel similar to a sandwich structure, and the testsample 302 is arranged on the flowcell.

In the present embodiment, referring to FIG. 11 , the device for imaging102 comprises a microscope 107 and a camera 108, the lens 104 comprisesa microscope objective 110 and a camera lens 112, and the focusingmodule 106 can be fixed with the camera lens 112 through a dichroic beamsplitter 114 which is located between the camera lens 112 and theobjective 110. The dichroic beam splitter 114 comprises a dual c-mountsplitter. The dichroic beam splitter 114 can reflect the light emittedby the focusing module 106 to the objective 110 and enable visible lightto pass through the objective and get into the camera 108 via the cameralens 112, as shown in FIG. 11 .

In an embodiment of the present disclosure, the lens 104 moves along theoptical axis OP. The movement of the lens 104 may refer to the movementof the objective 110, and the position of the lens 104 may refer to theposition of the objective 110. In other embodiments, other lenses of thelens 104 can be chosen to move to realize focusing. In addition, themicroscope 107 further comprises a tube lens 111 located between theobjective 110 and the camera 108. In the present embodiment, the carriercan drive a sample 200 to move on a plane (e.g., an XY plane)perpendicular to the direction of the optical axis OP (e.g., the Z axis)of the lens 104 and/or drive the sample 300 to move in the direction ofthe optical axis OP (e.g., the Z axis) of the lens 104.

In other embodiments, the plane on which the sample 300 is driven by thecarrier to move is not perpendicular to the optical axis OP, that is,the included angle between the movement plane of the sample and the XYplane is not 0, but the method for imaging is still applicable.

In addition, the device for imaging 102 can also drive the objective 110to move in the direction of the optical axis OP of the lens 104 forfocusing. In some embodiments, the device for imaging 102 drives theobjective 110 to move using a driving part such as a step motor or avoice coil motor.

In the present embodiment, when a coordinate system is established, asshown in FIG. 10 , the positions of the objective 110, the carrier andthe sample 300 can be provided on the negative axis of the Z axis, andthe first set position can be a coordinate position on the negative axisof the Z axis. It can be understood that in other embodiments, therelationship between the coordinate system and the camera and therelationship between the coordinate system and the objective 110 canalso be adjusted according to actual condition, which is notspecifically limited herein.

In one example, the device for imaging 102 comprises a total internalreflection fluorescent microscope, the magnification of the objective110 is 60×, and the first set step length S1 is equal to 0.01 mm. Thus,the first set step length S1 is suitable, because a too large first setstep length S1 will exceed an acceptable focusing range and a too smallfirst set step length S1 will increase time consumption.

When the focusing module 106 does not receive the light reflected by theobject, the lens 104 continues to move toward the sample 300 and theobject at the first set step length.

In the present embodiment, the system for imaging can be applied to asequence determination system, that is, the sequence determinationsystem comprises the system for imaging.

In the present embodiment, with the current position as a reference, thefirst range comprises a first interval and a second interval opposite toeach other, the second interval is defined closer to the sample, andstep (e) comprises: (i) when the second set position is located withinthe second interval, moving the lens from the second set position towarda direction away from the object, and acquiring an image of the objectat a position of each step using the device for imaging; or (ii) whenthe second set position is located within the first interval, moving thelens from the second set position toward a direction close to theobject, and acquiring an image of the object at the position of eachstep using the device for imaging. Thus, the movement of the lens can becontrolled according to the specific position of the second setposition, so that the desired image can be quickly acquired.

Specifically, in one example, a coordinate axis Z1 can be established inthe direction of the optical axis of the lens with the current positionas an origin oPos, the first interval is a positive interval, and thesecond interval is a negative interval. The range of the positive andnegative intervals is ±rLen, that is, the first range is [oPos+rLen,oPos−rLen]. The second set position is located within the negativeinterval and is (oPos−3×r0). r0 represents the second set step length.The device for imaging starts to perform image acquisition at(oPos−3×r0), and moves toward a direction away from the object.

It should be noted that the coordinate axis Z1 established in theaforementioned example is superposed with the Z axis in FIG. 10 , andthe first range is located within the negative interval of the Z axis.Thus, the control of the method for imaging can be simplified. Forexample, as long as the positional relationship between the origin ofthe Z axis and the origin oPos, the corresponding relationship betweenthe position of the lens on the coordinate axis Z1 and the position ofthe lens on the Z axis can be known.

In the present embodiment, step (f) comprises: comparing imageevaluation result with a preset condition, and if the image evaluationresult meets the preset condition, saving the position of the lens 104corresponding to the image; if the image evaluation result does not meetthe preset condition, moving the lens 104 to a third set position,wherein the third set position is located within another interval of thefirst range different from the interval where the second set position islocated, i.e., starting focusing for reverse photographing. For example,in the process of performing part (i) of step (e), none of the imageevaluation result meets the preset condition, and then the lens 104 ismoved to the third set position, namely moving the lens to the initialposition of performing part (ii) of step (e), and the focusing forreverse photographing, i.e., the process of performing part (ii) of step(e), is performed. Thus, searching a focusing position for an image inthe first range effectively increases the efficiency of the method forimaging.

Specifically, referring to the example of the aforementioned embodiment,the second set position is located within (oPos−3×r0) of the negativeinterval, the lens is moved upward from the second set position, and thedevice for imaging 102 acquires image at the position of each step; ifimage evaluation result does not meet the preset condition, then thelens 104 is moved to the third set position (e.g., (oPos+3×r0)) locatedwithin the positive interval; the device for imaging 102 then starts toacquire image from (oPos+3*r0) and moves toward a direction close to theobject, and according to the image evaluation result, focusing isrealized. When the image evaluation result meets the preset condition,the current position of the lens 104 corresponding to the image is savedas a saved position, which enable the device for imaging 102 to output aclear image when photographing in a sequence determination reaction.

In some embodiments, the image evaluation result comprises a firstevaluation value and a second evaluation value, the second set steplength comprises a rough step length and a fine step length, and step(f) comprises: moving the lens at the rough step length until the firstevaluation value of the image at the corresponding position is notgreater than a first threshold, moving the lens 104 at the fine steplength until the second evaluation value of the image at thecorresponding position is maximal, and saving the position of the lens104 corresponding to the image when the second evaluation value ismaximal Thus, the rough step length can make the lens 104 get close tothe focusing position quickly, and the fine step length can ensure thatthe lens 104 can reach the focusing position.

Specifically, the position of the lens 104 corresponding to the imagewith the maximal second evaluation value can be saved as a focusingposition. When the device for imaging 102 is used to acquire an image atthe position of each step, a first evaluation value and a secondevaluation value are calculated for the acquired image.

In one example, in the process of sequence determination, the object isprovided with an optically detectable label, such as a fluorescentlabel, and fluorescent molecules can be excited to emit fluorescenceunder the laser irradiation with a specific wavelength. The imageacquired by the device for imaging 102 comprises spots that maycorrespond to the positions of the fluorescent molecules. It can beunderstood that when the lens 104 is located at a focusing position, thespots corresponding to the positions of the fluorescent molecules in theacquired image are smaller and brighter; when the lens 104 is located ata non-focusing position, the spots corresponding to the positions of thefluorescent molecules in the acquired image are larger and less bright.

In the present embodiment, the image is evaluated by the size andintensity of the spots on the image.

For example, the first evaluation value is used to represent the size ofthe spot of the image. In one example, the first evaluation value isdetermined by calculating sizes of connected components corresponding tothe spots of the image, and pixels connectivity greater than the averagepixel value of the image is defined as a connected component. The firstevaluation value can be determined, for example, by calculating the sizeof the corresponding connected component of each spot, and the averagevalue of the sizes of the connected components corresponding to thespots is taken to represent a characteristic of the image as the firstevaluation value of the image. For another example, the sizes of theconnected components corresponding to the spots can be ranked from thesmallest size to the largest size, and the size of the connectedcomponent at the 50th, 60th, 70th, 80th or 90th quantile is taken as thefirst evaluation value of the image.

In one example, the size Area of a connected component corresponding toa spot of the image is equal to A×B with A representing the size of theconnected component in row centered on the center of the matrixcorresponding to the spot and B representing the size of the connectedcomponent in column centered on the center of the matrix correspondingto the spot. The matrix corresponding to the spot is defined as a matrixk1×k2 composed of odd rows and odd columns, which contains k1×k2 pixels.

In one example, the image is binarized firstly, and then the image isconverted into a digital matrix for calculating the size of connectedcomponents. For example, with the average pixel value of the image as areference, pixels not less than the average pixel value are marked as 1,and pixels less than the average pixel value are marked as 0, as shownin FIG. 12 . In FIG. 12 , the center of the matrix corresponding to thespot is shown in bold and enlarged, and a thick frame represents a 3×3matrix. The connected pixels marked as 1 form a connected component, andthe size of the connected component corresponding to the spot is A×B,wherein A×B=3×6.

The first threshold may be set according to experience or prior data. Inone example, the first evaluation value represents the size of the spotson the image. It is observed that the connected component Area firstlybecame smaller and then larger in the process of getting close to aclear surface and getting away from the clear surface. The firstthreshold is determined based on the numerical value and change rule ofArea in the focusing process where the clear surface is found multipletimes. In one example, the first threshold is set as 260. It should benoted that the first threshold can be related to the setting of therough step length and the fine step length: the numerical value of thefirst threshold cannot be such a value that the focal plane for theimaging of the object by the device for imaging is passed in only onerough step length.

In some embodiments, the second evaluation value or the third evaluationvalue is determined by calculating scores of the spots of the image, andthe score of the spot of one image is equal to((k1×k2−1)CV−EV)/((CV+EV)/(k1×k2)), wherein CV represents the centralpixel value of the matrix corresponding to the spot, and EV representsthe sum of the non-central pixel values of the matrix corresponding tothe spot. Thus, the second evaluation value or the third evaluationvalue can be determined.

Specifically, after the spots of the image are determined, the Scorevalues of all the spots of the image can be ranked in an ascendingorder. When the number of the spots is greater than a preset number, forexample, the preset number is 30 and the number of the spots is 50, thesecond evaluation value may be a Score value at the 50th, 60th, 70th,80th or 90th quantile, and thus, the interference of 50%, 60%, 70%, 80%or 90% of the spots with relatively poor quality can be eliminatedGenerally, it is considered that the intensity/pixel value differencebetween the center and edge is great and that the converged spots arespots corresponding to the molecules to be detected. The molecules to bedetected may represent nucleic acid molecules which correspond to targetdetected objects during nucleic acid detection.

When the number of the spots is less than the preset number, forexample, the number of the spots is 10 and less than the preset number,the number of spots is such small that it is not statisticallysignificant. Therefore, the spot with the largest Score value is takento represent the image, that is, a 100th quantile Score value is takenas the third evaluation value.

In the present embodiment, the image evaluation result comprises a firstevaluation value, a second evaluation value, and a third evaluationvalue, and the image comprises a plurality of pixels; the presetcondition is: the number of spots on the image is greater than a presetvalue, the first evaluation value of the image at a correspondingposition is not greater than the first threshold, and the secondevaluation value of the image at the corresponding position is maximalamong the second evaluation values of N images before and after theimage at the corresponding position; or the preset condition is: thenumber of spots on the image is less than the preset value, the firstevaluation value of the image at the corresponding position is notgreater than the first threshold, and the third evaluation value of theimage at the corresponding position is maximal among the thirdevaluation values of N images before and after the current image. Thus,by evaluating with different evaluation values according to the numberof the spots of the image, the focusing of the method for imaging ismore accurate.

Specifically, in one example, the first evaluation value may be the sizeof a connected component corresponding to a spot of the image in theaforementioned embodiment. The second evaluation value and the thirdevaluation value are different Score quantiles taken according towhether the number of spots is statistically significant or not indifferent examples. For example, the second evaluation value and thethird evaluation value may be respectively a non-100th quantile Scorevalue and a 100th quantile Score value.

In one example, single-molecule sequencing is performed, and the spotson the acquired image may come from one or several optically detectablelabeled molecules carried by test samples or from other interference.

In the present embodiment, the spots are detected, that is, the spotscorresponding to/coming from the labeled molecules are detected. Forexample, the spots can be detected using a k1×k2 matrix. Specifically,the following method is used to detect the spots on the image:

using k1×k2 matrices to detect the spots on the image, comprisingdetermining that the matrix in which a central pixel value of the matrixis not less than any non-central pixel value of the matrix correspondsto a spot, wherein both k1 and k2 are odd numbers greater than 1, andthe k1×k2 matrix comprises k1×k2 pixels.

Based on the difference between brightness/intensity of signalsgenerated by fluorescence and background brightness/intensity, themethod can simply and quickly detect information coming from labeledmolecule signals. In some embodiments, the central pixel value of thematrix is greater than a first preset value, and any non-central pixelvalue of the matrix is greater than a second preset value.

The first preset value and the second preset value may be set accordingto experience or pixel/intensity data of normal spots of a certainnumber of normal images. The “normal image” and “normal spot” may be animage acquired by the system for imaging at a position of clear surfaceand looks normal to the naked eyes, for example, the image looks clearwith clean background, uniform spot size and brightness, etc. In oneembodiment, the first preset value and the second preset value arerelated to the average pixel value of the image. For example, the firstpreset value is set as 1.4 times the average pixel value of the image,the second preset value is set as 1.1 times the average pixel value ofthe image, and therefore interference can be eliminated and a spotdetection result can be acquired from labels.

Specifically, in one example, the image is a color image having threepixel values for each pixel and the color image may be converted into agrayscale image before image detection, so as to reduce the calculationand complexity in an image detection process. A non-grayscale image maybe converted into a grayscale image with methods including but notlimited to floating point algorithm, integer method, shift method, meanvalue method. Certainly, the color image may also be directly detected,the comparison of the aforementioned pixel values may be regarded as thecomparison of three-dimensional values or arrays with three elements,and the relative values of a plurality of multi-dimensional values canbe customized according to experience and need. For example, when anytwo-dimensional numerical values in a three-dimensional value a aregreater than the corresponding-dimensional numerical values in athree-dimensional value b, it can be considered that the a is greaterthan the b.

In another example, the image is a grayscale image, the pixel value ofwhich is a grayscale value. Therefore, the average pixel value of theimage is average grayscale value of the image.

In one example, the first threshold is 260, the preset number is 30, andN is equal to 2. That is, when the first evaluation value of an image ata corresponding position is not greater than 260 and the number of spotsis greater than 30, the second evaluation value of the image at thecorresponding position is statistically acquired, the position of theimage with the maximal second evaluation value is determined as theposition of a clear surface, and two positions meeting the followingcondition exist before and after the position: the second evaluationvalue of the corresponding image is greater than zero. When the firstevaluation value of an image at a corresponding position is not greaterthan 260 and the number of spots is less than 30, the third evaluationvalue of the image at the corresponding position is statisticallyacquired, the position of the image with the maximal third evaluationvalue is determined as the position of a clear surface, and twopositions meeting the following condition exist before and after theposition: the third evaluation value of the corresponding image isgreater than zero.

If no image meeting the aforementioned condition is found, it isdetermined that the image evaluation result does not meet the presetcondition.

In one example, k1=k2=3, then there are nine pixels in a 3×3 matrix, andEV is the sum of eight non-central pixel values.

In the present embodiment, if focusing cannot be realized according tothe image evaluation result, the lens is moved to the next imageacquisition region (FOV) in a direction perpendicular to the opticalaxis for focusing. Thus, refocusing can be performed from other objects,avoiding constant focusing on the current object on which focusingfails, thus saving time.

In the present embodiment, the method for imaging further comprises:when the number of the current objects on which focusing fails isgreater than a preset number, a prompt is given for focusing failure.Thus, a cause of focusing failure can be eliminated manually to avoidconstant focusing, thus saving time. Specifically, in this case, thecause may be the wrong placements of the objects or the failure of thedevice for imaging. After a prompt for focusing failure is given, thecause of focusing failure can be eliminated manually. In one example,the preset number is 3, that is, when the number of the current objectson which focusing fails is greater than 3, a prompt is given forfocusing failure. The way to give a prompt for focusing failure may bedisplaying an image or words, playing sound, etc.

In the present embodiment, the method for imaging further comprises:determining whether the position of a lens exceeds a first range, andquitting focusing when the position of the lens exceeds the first range.Thus, quitting focusing when the position of the lens exceeds the firstrange can prevent too long focusing time and an increase in powerconsumption.

Specifically, in the example of the aforementioned embodiment, the firstrange is [oPos+rLen, oPos−rLen].

In the present embodiment, as lens 104 moves, whether the currentposition of the lens 104 is beyond a fourth set position is determined;and when the current position of the lens 104 is beyond the fourth setposition, the movement of the lens 104 is stopped. Thus, the first setposition and the fourth set position can define the movement range (thefirst range) of the lens 104, so that the lens 104 can stop moving whenfocusing fails, preventing resource waste or equipment damage, or thelens 104 can refocus when focusing fails, increasing the automationdegree of the method for imaging.

For example, in a total internal reflection system for imaging, in orderto quickly find a medium interface, setting will be adjusted to make themovement range of the lens 104 as small as possible under the conditionthat the solution can be implemented. For example, on a total internalreflection device for imaging with a 60× objective, the movement rangeof the lens 104 may be set as 200 μm±10 μm or [190 μm, 250 μm] accordingto characteristics of optical path and experience.

In the present embodiment, another set position can be determinedaccording to the setting of the determined movement range and any of thefourth set position and the first set position. In one example, thefourth set position is set as the position a depth of field lower thanthe lowest position of an upper surface 205 of the front panel 202 ofthe reaction device 200, the movement range of the lens 104 is set as250 μm, and thus, the first set position is determined.

In the example of the present disclosure, the coordinate positioncorresponding to the position a depth of field lower is a position whichbecomes smaller along the negative direction of the Z axis.

Specifically, in the present embodiment, the movement range is aninterval on the negative axis of the Z axis. In one example, the firstset position is nearlimit, the fourth set position is farlimit, and thecoordinate positions corresponding to nearlimit and farlimit are locatedon the negative axis of the Z axis; nearlimit is equal to −6000 um, andfarlimit is equal to −6350 um. The movement range defined betweennearlimit and farlimit is 350 um. Therefore, when the coordinateposition corresponding to the current position of the lens 104 is lessthan the coordinate position corresponding to the fourth set position,it is determined that the current position of the lens 104 is beyond thefourth set position. In FIG. 10 , the position of farlimit is theposition a depth of field L lower than the lowest position of the uppersurface 205 of the front panel 202 of the reaction device 200. The depthof field L is the depth of field of the lens 104.

It should be noted that, in other embodiments, the coordinate positionscorresponding to the first set position and/or the fourth set positionmay be specifically set according to actual condition, which is notspecifically limited herein.

In the present embodiment, the focusing module 106 comprises a lightsource 116 and an optical sensor 118, the light source 116 is configuredfor emitting light onto an object, and the optical sensor 118 isconfigured for receiving the light reflected by the object. Thus, thefocusing module 106 can emit and receive light.

Specifically, in the embodiment of the present disclosure, the lightsource 116 may be an infrared light source 116, and the optical sensor118 may be a photodiode. Therefore, the cost is low, and the accuracy ofdetection is high. Infrared light emitted by the light source 116 isreflected by a dichroic beam splitter to get into the objective 110, andis projected onto the sample 300 and an object by the objective 110. Theobject can reflect the infrared light projected by the objective 110. Inthe embodiment of the present disclosure, when the sample 300 comprisesthe carrying device 200 and the test sample 302, the received lightreflected by the object is the light reflected by the lower surface 204of the front panel of the carrying device 200.

Whether the infrared light reflected by the object can get into theobjective 110 and be received by the optical sensor 118 mainly dependson the distance between the objective 110 and the object. Therefore,when it is determined that the focusing module 106 receives the infraredlight reflected by the object, it can be determined that the distancebetween the objective 110 and the object is within an appropriateoptical imaging range and can be used for imaging by the device forimaging 102. In one example, the distance is 20 um to 40 um.

At this point, the lens 104 is moved at the second set step length whichis smaller than the first set step length, so that the system forimaging can find the best imaging position of the lens 104 in a smallerrange.

In the present embodiment, referring to FIG. 13 , when the focusingmodule 106 receives the light reflected by the object, the method forimaging further comprises: (g) moving the lens 104 toward the object ata third set step length less than the first set step length and greaterthan the second set step length, calculating a first light intensityparameter according to the light intensity of the light received by thefocusing module 106, and determining whether the first light intensityparameter is greater than a first set light intensity threshold; andwhen the first light intensity parameter is greater than the first setlight intensity threshold, performing step (d). Thus, by comparing thefirst light intensity parameter with the first set light intensitythreshold, the interference of light signals much weaker than the lightreflected by the medium interface on focusing can be eliminated.

When the first light intensity parameter is not greater than the firstset light intensity threshold, the lens 104 continues to be moved towardthe object at the third set step length.

In the present embodiment, the focusing module 106 comprises two opticalsensors 118, the two optical sensors 118 are configured for receivingthe light reflected by the object, and the first light intensityparameter is an average value of light intensities of light received bythe two optical sensors 118. Thus, the first light intensity parameteris calculated by the average value of light intensities of lightreceived by the two optical sensors 118, so that weak light signals canbe more accurately eliminated.

Specifically, the first light intensity parameter may be set as SUM,i.e., SUM=(PD1+PD2)/2, and PD1 and PD2 respectively represent lightintensities of light received by the two optical sensors 118. In oneexample, the first set light intensity threshold nSum is equal to 40.

In one example, the third set step length S2 is equal to 0.005 mm. Itcan be understood that, in other examples, the third set step length mayalso be other numerical values, which are not specifically definedherein.

Embodiment 2

It should be noted that, in the present embodiment, the structuraldiagram of the system for imaging in embodiment 1 can be adopted as thestructural diagram of the system for imaging. It can be understood thatthe focusing method in embodiment 2 is different from the focusingmethod or focusing logic in embodiment 1, but the structures of thesystems for imaging used are basically the same.

Referring to FIGS. 10, 11 and 14 , focusing comprises the followingsteps: (S11) emitting light onto an object using the focusing module106; (S12) moving the lens 104 to a first set position; (S13) moving thelens 104 from the first set position toward the object at a first setstep length and determining whether the focusing module 106 receives thelight reflected by the object; (S14) when the focusing module 106receives the light reflected by the object, moving the lens 104 at asecond set step length smaller than the first set step length, acquiringan image of the object using the device for imaging 102, and determiningwhether the sharpness value of the image acquired by the device forimaging 102 reaches a set threshold; and (S15) when the sharpness valueof the image reaches the set threshold, saving the current position ofthe lens 104 as a saved position.

Using the aforementioned focusing method, a plane for clear imaging of atarget object, i.e., a clear plane/clear surface, can be quickly andaccurately found. The method is particularly suitable for a devicecomprising a precision optical system and having difficulty in finding aclear plane, such as an optical detection device with ahigh-magnification lens.

Specifically, in the aforementioned focusing step, the object is anobject whose focal plane position needs to be acquired. For example, ifa first predetermined relationship needs to be determined, two objectscan be selected on a first preset track, and focusing is successively orsimultaneously performed on the two objects on the first preset track 43to acquire two sets of focal plane position data, wherein one set offocal plane position data serves as the focal plane position data of thefirst object 42 and the other serves as the focal plane position data ofthe second object 44; if a second predetermined relationship needs to bedetermined, two objects can be selected on a second preset track,focusing can be successively or simultaneously performed on the twoobjects located on the second preset track 45 to acquire two sets offocal plane position data, wherein one set of focal plane position dataserves as the focal plane position data of the fourth object 47 and theother serves as the focal plane position data of the fifth object 48.

Referring to FIG. 10 , in the embodiment of the present disclosure,objects are a plurality of positions (FOVs) of a sample 300 applied insequence determination. Specifically, when the first predeterminedrelationship needs to be determined, the object for focusing may serveas a first object or a second object; and when the second predeterminedrelationship needs to be determined, the object for focusing may serveas a fourth object or a fifth object. The sample 300 comprises acarrying device 200 and a test sample 302 located on the carryingdevice. The test sample 302 is a biomolecule, such as a nucleic acid.The lens 104 is over the carrying device 200. The carrying device 200 isprovided with a front panel 202 and a back panel (lower panel), each ofwhich is provided with two surfaces, and the test sample 302 isconnected to the upper surface of the lower panel, that is, the testsample 302 is located under the lower surface 204 of the front panel202. In an embodiment of the present disclosure, because the device forimaging 102 acquires an image of the test sample 302, the test sample302 is the corresponding position (FOV) during photographing. The testsample 302 is located under the lower surface 204 of the front panel 202of the carrying device 200, and when a focusing process begins, the lens104 moves in order to find a medium interface 204 where the test sample302 is located so as to increase success rate of the device for imaging102 in acquiring a clear image. In an embodiment of the presentdisclosure, the test sample 302 is a solution, the front panel 202 ofthe carrying device 200 is glass, and the medium interface 204 betweenthe carrying device 200 and the test sample 302 is the lower surface 204of the front panel 202 of the carrying device 200, i.e., the interfacebetween two media (glass and liquid). The test sample 302 from which thedevice for imaging 102 needs to acquire an image is located under thelower surface 204 of the front panel 202, and at this time, a clearsurface for the clear imaging of the test sample 302 is determined andfound by the image acquired by the device for imaging 102. This processcan be referred to as focusing. In one example, the thickness of thefront panel 202 is 0.175 mm.

In the present embodiment, the carrying device 200 may be a slide, andthe test sample 302 is placed on the slide or the test sample 302 isclamped between two slides. In some embodiments, the carrying device 200may be a reaction device, e.g., a flowcell with an upper carrying paneland a lower carrying panel similar to a sandwich structure, and the testsample 302 is arranged on the flowcell.

In the present embodiment, referring to FIG. 11 , the device for imaging102 comprises a microscope 107 and a camera 108, the lens 104 comprisesa microscope objective 110 and a camera lens 112, and the focusingmodule 106 can be fixed with the camera lens 112 through a dichroic beamsplitter 114 which is located between the camera lens 112 and theobjective 110. The dichroic beam splitter 114 comprises a dual c-mountsplitter. The dichroic beam splitter 114 can reflect the light emittedby the focusing module 106 to the objective 110 and enable visible lightto pass through the objective and get into the camera 108 via the cameralens 112, as shown in FIG. 11 .

In an embodiment of the present disclosure, the lens 104 moves along theoptical axis OP. The movement of the lens 104 may refer to the movementof the objective 110, and the position of the lens 104 may refer to theposition of the objective 110. In other embodiments, other lenses of thelens 104 can be chosen to move to realize focusing. In addition, themicroscope 107 further comprises a tube lens 111 located between theobjective 110 and the camera 108. In the present embodiment, the carriercan drive a sample 200 to move on a plane (e.g., an XY plane)perpendicular to the optical axis OP (e.g., the Z axis) of the lens 104and/or drive a sample 300 to move along the optical axis OP (e.g., the Zaxis) of the lens 104.

In other embodiments, the plane on which the sample 300 is driven by thecarrier to move is not perpendicular to the optical axis OP, that is,the included angle between the movement plane of the sample and the XYplane is not 0, but the method for imaging is still applicable.

In addition, the device for imaging 102 can also drive the objective 110to move along the optical axis OP of the lens 104 for focusing. In someembodiments, the device for imaging 102 drives the objective 110 to moveusing a driving part such as a step motor or a voice coil motor.

In the present embodiment, when a coordinate system is established, asshown in FIG. 10 , the positions of the objective 110, the carrier andthe sample 300 can be provided on the negative axis of the Z axis, andthe first set position can be a coordinate position on the negative axisof the Z axis. It can be understood that in other embodiments, therelationship between the coordinate system and the camera and therelationship between the coordinate system and the objective 110 canalso be adjusted according to actual condition, which is notspecifically limited herein.

In one example, the device for imaging 102 comprises a total internalreflection fluorescent microscope, the magnification of the objective110 is 60×, and the first set step length S1 is equal to 0.01 mm. Thus,the first set step length S1 is suitable, because a too large first setstep length S1 will exceed an acceptable focusing range and a too smallfirst set step length S1 will increase time consumption.

When the focusing module 106 does not receive the light reflected by theobject, the lens 104 continues to be moved toward the sample 300 and theobject at the first set step length along the optical axis OP.

In the present embodiment, when the sharpness value of the image doesnot reach the set threshold, the lens 104 continues to be moved alongthe optical axis OP at the second set step length.

In the present embodiment, the system for imaging can be applied to asequence determination system, that is, the sequence determinationsystem comprises the system for imaging.

In the present embodiment, as lens 104 moves, whether the currentposition of the lens 104 is beyond a second set position is determined;and when the current position of the lens 104 is beyond the second setposition, the movement of the lens 104 is stopped or the focusing stepis performed. Thus, the first set position and the second set positioncan define the movement range of the lens 104, so that the lens 104 canstop moving when focusing fails, preventing resource waste or equipmentdamage, or the lens 104 can refocus when focusing fails, increasing theautomation degree of the method for imaging.

For example, in a total internal reflection system for imaging, in orderto quickly find a medium interface, setting will be adjusted to make themovement range of the lens 104 as small as possible under the conditionthat the solution can be implemented. For example, on a total internalreflection device for imaging with a 60× objective, the movement rangeof the lens 104 may be set as 200 μm±10 μm or [190 μm, 250 μm] accordingto characteristics of optical path and experience.

In the present embodiment, another set position can be determinedaccording to the setting of the determined movement range and any of thesecond set position and the first set position. In one example, thesecond set position is set as the position a depth of field lower thanthe lowest position of an upper surface 205 of the front panel 202 ofthe reaction device 200, the movement range of the lens 104 is set as250 μm, and thus, the first set position is determined. In the exampleof the present disclosure, the coordinate position corresponding to theposition a depth of field lower is a position which becomes smalleralong the negative direction of the Z axis.

Specifically, in the embodiment of the present disclosure, the movementrange is an interval on the negative axis of the Z axis. In one example,the first set position is nearlimit, the second set position isfarlimit, and the coordinate positions corresponding to nearlimit andfarlimit are located on the negative axis of the Z axis; nearlimit isequal to −6000 um, and farlimit is equal to −6350 um. The movement rangedefined between nearlimit and farlimit is 350 um. Therefore, when thecoordinate position corresponding to the current position of the lens104 is less than the coordinate position corresponding to the second setposition, it is determined that the current position of the lens 104 isbeyond the second set position. In FIG. 10 , the position of farlimit isthe position a depth of field L lower than the lowest position of theupper surface 205 of the front panel 202 of the reaction device 200. Thedepth of field L is the depth of field of the lens 104.

It should be noted that, in other embodiments, the coordinate positionscorresponding to the first set position and/or the second set positionmay be specifically set according to actual condition, which is notspecifically limited herein.

In the present embodiment, the focusing module 106 comprises a lightsource 116 and an optical sensor 118, the light source 116 is configuredfor emitting light onto an object, and the optical sensor 118 isconfigured for receiving the light reflected by the object. Thus, thefocusing module 106 can emit and receive light.

Specifically, in the embodiment of the present disclosure, the lightsource 116 may be an infrared light source 116, and the optical sensor118 may be a photodiode. Therefore, the cost is low, and the accuracy ofdetection is high. Infrared light emitted by the light source 116 isreflected by a dichroic beam splitter to get into the objective 110, andis projected onto the sample 300 and an object by the objective 110. Theobject can reflect the infrared light projected by the objective 110. Inthe embodiment of the present disclosure, when the sample 300 comprisesthe carrying device 200 and the test sample 302, the received lightreflected by the object is the light reflected by the lower surface 204of the front panel of the carrying device 200.

Whether the infrared light reflected by the object can get into theobjective 110 and be received by the optical sensor 118 mainly dependson the distance between the objective 110 and the object. Therefore,when it is determined that the focusing module 106 receives the infraredlight reflected by the object, it can be determined that the distancebetween the objective 110 and the object is within an appropriateoptical imaging range and can be used for imaging by the device forimaging 102. In one example, the distance is 20 um to 40 um.

At this point, the lens 104 is moved at the second set step length whichis smaller than the first set step length, so that the system forimaging can find the best imaging position of the lens 104 in a smallerrange.

In the present embodiment, the sharpness value of the image can serve asan evaluation value of image focusing. In one example, whether thesharpness value of the image acquired by the device for imaging 102reaches the set threshold can be determined by the hill climbingalgorithm for image processing. Whether the sharpness value reaches amaximal value at a sharpness value peak is determined by calculating thesharpness value of the image outputted by the device for imaging 102when the objective 110 is at each position, and whether the lens 104reaches the position of a clear surface during imaging by the device forimaging 102 is then determined. It can be understood that, in otherembodiments, other algorithms for image processing can also be used todetermine whether the sharpness value reaches the maximal value at thepeak.

When the sharpness value of the image reaches the set threshold, thecurrent position of the lens 104 is saved as a saved position, whichenable the device for imaging 102 to output a clear image whenphotographing in a sequence determination reaction.

In the present embodiment, referring to FIG. 15 , when the focusingmodule 106 receives the light reflected by the object, focusing furthercomprises: (S16) moving the lens 104 toward the object at a third setstep length less than the first set step length and greater than thesecond set step length, calculating a first light intensity parameteraccording to the light intensity of the light received by the focusingmodule 106, and determining whether the first light intensity parameteris greater than a first set light intensity threshold; and when thefirst light intensity parameter is greater than the first set lightintensity threshold, performing step (S14). Thus, by comparing the firstlight intensity parameter with the first set light intensity threshold,the interference of light signals much weaker than the light reflectedby the medium interface on focusing can be eliminated.

When the first light intensity parameter is not greater than the firstset light intensity threshold, the lens 104 continues to be moved towardthe object at the third set step length along the optical axis OP.

In the present embodiment, the focusing module 106 comprises two opticalsensors 118, the two optical sensors 118 are configured for receivingthe light reflected by an object, and the first light intensityparameter is an average value of light intensities of light received bythe two optical sensors 118. Thus, the first light intensity parameteris calculated by the average value of light intensities of lightreceived by the two optical sensors 118, so that weak light signals canbe more accurately eliminated.

Specifically, the first light intensity parameter may be set as SUM,i.e., SUM=(PD1+PD2)/2, and PD1 and PD2 respectively represent lightintensities of light received by the two optical sensors 118. In oneexample, the first set light intensity threshold nSum is equal to 40.

In one example, the third set step length S2 is equal to 0.005 mm. Itcan be understood that, in other examples, the third set step length mayalso be other numerical values, which are not specifically definedherein.

In another embodiment, referring to FIG. 16 , when the focusing module106 receives the light reflected by the object, the imaging methodfurther comprises: (S16) moving the lens 104 toward the object at athird set step length less than the first set step length and greaterthan the second set step length, calculating a first light intensityparameter according to the light intensity of the light received by thefocusing module 106, and determining whether the first light intensityparameter is greater than a first set light intensity threshold; (S17)when the first light intensity parameter is greater than the first setlight intensity threshold, moving the lens 104 toward the object at afourth set step length less than the third set step length and greaterthan the second set step length, calculating a second light intensityparameter according to the light intensity of the light received by thefocusing module 106, and determining whether the second light intensityparameter is less than a second set light intensity threshold; and whenthe second light intensity parameter is less than the second set lightintensity threshold, performing step (S14). Thus, by comparing the firstlight intensity parameter with the first set light intensity threshold,the interference of light signals much weaker than the light reflectedby the medium interface on focusing can be eliminated; and by comparingthe second light intensity parameter with the second set light intensitythreshold, the interference of strongly reflected light signals not fromthe position of the medium interface (e.g., light signals reflected bythe oil surface of the objective 110/air) on focusing can be eliminated.

When the first light intensity parameter is not greater than the firstset light intensity threshold, the lens 104 continues to be moved towardthe object at the third set step length along the optical axis OP.

When the second light intensity parameter is not less than the secondset light intensity threshold, the lens 104 continues to be moved towardthe object at the fourth set step length along the optical axis OP.

In one example, the third set step length S2 is equal to 0.005 mm, andthe fourth set step length S3 is equal to 0.002 mm. It can be understoodthat, in other examples, the third set step length and the fourth setstep length may also be other numerical values, which are notspecifically defined herein.

In the present embodiment, the focusing module 106 comprises two opticalsensors 118, the two optical sensors 118 are configured for receivingthe light reflected by the object, and the first light intensityparameter is an average value of the light intensities of the lightreceived by the two optical sensors 118; the light intensities of thelight received by the two optical sensors 118 have a first difference,and the second light intensity parameter is a difference between thefirst difference and a set compensated value. Thus, the second lightintensity parameter is calculated by the light intensities of the lightreceived by the two optical sensors 118, so that strongly reflectedlight signals can be more accurately eliminated.

Specifically, the first light intensity parameter may be set as SUM,i.e., SUM=(PD1+PD2)/2, and PD1 and PD2 respectively represent lightintensities of light received by the two optical sensors 118. In oneexample, the first set light intensity threshold nSum is equal to 40.The difference may be set as err, the set compensated value is offset,that is, err=(PD1−PD2)−offset. In an ideal state, the first differencemay be zero. In one example, the second set light intensity thresholdnErr is equal to 10, and offset is equal to 30.

In the present embodiment, when the lens 104 is moved at the second setstep length, whether the first sharpness value of the imagecorresponding to the current position of the lens 104 is greater thanthe second sharpness value of the image corresponding to the previousposition of the lens 104 is determined; when the first sharpness valueis greater than the second sharpness value and the sharpness differencebetween the first sharpness value and the second sharpness value isgreater than a set difference, the lens 104 continues to be moved towardthe object at the second set step length; when the first sharpness valueis greater than the second sharpness value and the sharpness differencebetween the first sharpness value and the second sharpness value is lessthan the set difference, the lens 104 continues to be moved toward theobject at a fifth set step length less than the second set step length,so that the sharpness value of the image acquired by the device forimaging 102 reaches the set threshold; when the second sharpness valueis greater than the first sharpness value and the sharpness differencebetween the second sharpness value and the first sharpness value isgreater than the set difference, the lens 104 is moved away from theobject at the second set step length; and when the second sharpnessvalue is greater than the first sharpness value and the sharpnessdifference between the second sharpness value and the first sharpnessvalue is less than the set difference, the lens 104 is moved away fromthe object at the fifth set step length, so that the sharpness value ofthe image acquired by the device for imaging 102 reaches the setthreshold. Thus, the position of the lens 104 corresponding to thesharpness value peak can be accurately found, so that the imageoutputted by the device for imaging is clear.

Specifically, the second set step length can serve as a rough adjustmentstep length Z1, the fifth set step length can serve as a fine adjustmentstep length Z2, and a rough adjustment range Z3 can be set. The settingof the rough adjustment range Z3 can stop the movement of the lens 104when the sharpness value of the image cannot reach the set threshold,thus saving resources.

With the current position of the lens 104 as a starting point T, therough adjustment range Z3 is an adjustment range, that is, theadjustment range on the Z axis is (T, T+Z3). The lens 104 is firstlymoved in a first direction (such as a direction approaching the objectalong the optical axis OP) within the range of (T, T+Z3) at the steplength Z1, and the first sharpness value R1 of the image acquired by thedevice for imaging 102 at the current position of the lens 104 iscompared with the second sharpness value R2 of the image acquired by thedevice for imaging 102 at the previous position of the lens 104.

When R1>R2 and R1−R2>R0, it means that the sharpness value of the imageis approaching the set threshold and is far from the set threshold, andtherefore the lens 104 continues to move in the first direction at thestep length Z1 to quickly approach the set threshold.

When R1>R2 and R1−R2<R0, it means that the sharpness value of the imageis approaching the set threshold and is close to the set threshold, andtherefore the lens 104 moves in the first direction at the step lengthZ2 to approach the set threshold at a smaller step length.

When R2>R1 and R2−R1>R0, it means that the sharpness value of the imagehas exceeded the set threshold and is far from the set threshold, andtherefore the lens 104 moves in a second direction (such as a directionaway from the object along the optical axis OP) opposite to the firstdirection at the step length Z1 to quickly approach the set threshold.

When R2>R1 and R2−R1<R0, it means that the sharpness value of the imagehas exceeded the set threshold and is close to the set threshold, andtherefore the lens 104 moves in the second direction opposite to thefirst direction at the step length Z2 to approach the set threshold at asmaller step length.

In the present embodiment, as the lens 104 moves, the fifth set steplength can be adjusted to adapt to the condition that the step lengthshould not be too large or too small when approaching the set threshold.

In one example, T=0, Z1=100, Z2=40, Z3=2100, and the adjustment range is(0, 2100). It should be noted that the aforementioned values are metricvalues used when the lens 104 is moved in the process of imageacquisition by the device for imaging 102, and the metric values arerelated to light intensity. The set threshold can be interpreted as apeak value of the focusing curve, a range with the peak as a center, ora range including the peak value.

Referring to FIG. 5 , a system for imaging 100 according to theembodiment of the present disclosure is configured for imaging objects,wherein the system for imaging comprises a lens 104 and a control device101, the objects comprise a first object 42, a second object 44 and athird object 46 located at different positions on a first preset track43, the control device 101 comprises a computer-executable program, andthe execution of the computer-executable program comprises the steps ofthe method for imaging according to any of the aforementionedembodiments.

In the aforementioned system for imaging 100, the first predeterminedrelationship is determined by the focusing positions of the first object42 and the second object 44, and when other objects on the first presettrack are imaged, focal planes can be directly predicted according tothe first predetermined relationship to acquire a clear image of thethird object without focusing. The system for imaging is particularlysuitable for a scenario where there are a lot of objects and it isdesired to quickly and continuously acquire images of these objects. Thesystem is high in imaging efficiency and is able to accurately determinethe focal plane positions of the subsequent objects to acquire the imageinformation of the subsequent objects in continuous image acquisitioneven if the system for imaging fails to track the focus. The system usedin cooperation with a focus tracking system of the system for imagingcan provide a remedy in the event that the focus tracking system cannotnormally track the focus again after it fails to track the focus.

It should be noted that the explanation and description of the technicalfeatures and benefits of the method for imaging in any of theaforementioned embodiments and examples are also applicable to thesystem for imaging 100 according to the present embodiment. Therefore,there will be no detailed description herein to avoid redundancy.

In some embodiments, the third object 46 is located between the firstobject 42 and the second object 44.

In some embodiments, the lens 104 is fixed and comprises an optical axisOP, and the first preset track 43 can move in a direction perpendicularor parallel to the optical axis OP.

In some embodiments, the determination of the first predeterminedrelationship comprises: focusing on the first object 42 using the systemfor imaging to determine first coordinates; focusing on the secondobject 44 using the system for imaging to determine second coordinates;and establishing the first predetermined relationship according to thefirst coordinates and the second coordinates, wherein the firstcoordinates represent a focal plane position of the first object 42, andthe second coordinates represent a focal plane position of the secondobject 44.

In some embodiments, the first preset track 43 is a linear or non-lineartrack; and/or the first predetermined relationship is a linearrelationship.

In some embodiments, the objects comprise a fourth object 47 and a fifthobject 48 located at different positions on a second preset track 45,and the control device 101 is configured for:

allowing the lens 104 and the second preset track 45 to move relativelyin a second predetermined relationship to acquire an image of the fifthobject 48 using the system for imaging without focusing, wherein thesecond predetermined relationship is determined by a focal planeposition of the fourth object 47 and the first predeterminedrelationship, and the second preset track 45 is different from the firstpreset track 43.

In some embodiments, the lens 104 is fixed and comprises an optical axisOP, and the second preset track 45 can move in a direction perpendicularor parallel to the optical axis OP.

In some embodiments, the determination of the second predeterminedrelationship comprises: focusing on the fourth object 47 using thesystem for imaging to determine fourth coordinates; and establishing thesecond predetermined relationship according to the first predeterminedrelationship and the fourth coordinates, wherein the fourth coordinatesrepresent a focal plane position of the fourth object 47.

In some embodiments, the control device 101 is configured for: allowing,after acquiring the image of the third object 46, the lens 104 and thefirst preset track 43 and/or the second preset track 45 to moverelatively to acquire an image of the fifth object 48 using the systemfor imaging without focusing.

In some embodiments, the system for imaging comprises a device forimaging 102 and a carrier 103. The device for imaging 102 comprises alens 104 and a focusing module 106. The lens 104 comprises an opticalaxis OP, and the lens 104 can move in the direction of the optical axisOP. A first preset track 43 and/or a second preset track 45 are locatedon the carrier 103.

In some embodiments, the control device 101 is configured for performingthe following steps: (a) emitting light onto the object using thefocusing module 106; (b) moving the lens 104 to a first set position;(c) moving the lens 104 from the first set position toward the object ata first set step length and determining whether the focusing module 106receives the light reflected by the object; (d) when the focusing module106 receives the light reflected by the object, moving the lens 104 fromthe current position to a second set position, wherein the second setposition is located within a first range, and the first range is a rangeincluding the current position and allowing the lens 104 to move in thedirection of the optical axis OP; (e) moving the lens 104 from thesecond set position at a second set step length and acquiring an imageof the object at a position of each step using the device for imaging102, wherein the second set step length is less than the first set steplength; and (f) evaluating the images of the object to obtain an imageevaluation result, and focusing according to the image evaluationresult.

In some embodiments, with the current position as a reference, the firstrange comprises a first interval and a second interval opposite to eachother, the second interval is defined closer to the object, and step (e)comprises: (i) when the second set position is located within the secondinterval, moving the lens 104 from the second set position toward adirection away from the object and acquiring an image of the object at aposition of each step using the device for imaging 102; or (ii) when thesecond set position is located within the first interval, moving thelens 104 from the second set position toward a direction close to theobject and acquiring an image of the object at a position of each stepusing the device for imaging 102.

In some embodiments, step (f) comprises: comparing image evaluationresult with a preset condition, and if the image evaluation result meetsthe preset condition, saving the position of the lens 104 correspondingto the image; if the image evaluation result does not meet the presetcondition, moving the lens 104 to a third set position, wherein thethird set position is located within another interval of the first rangedifferent from the interval where the second set position is located.

In some embodiments, the image evaluation result comprises a firstevaluation value and a second evaluation value, the second set steplength comprises a rough step length and a fine step length, and step(f) comprises: moving the lens 104 at the rough step length until thefirst evaluation value of the image at the corresponding position is notgreater than a first threshold, moving the lens 104 at the fine steplength until the second evaluation value of the image at thecorresponding position is maximal, and saving the position of the lens104 corresponding to the image when the second evaluation value ismaximal.

In some embodiments, the image evaluation result comprises a firstevaluation value, a second evaluation value and a third evaluationvalue, and the image comprises a plurality of pixels; the presetcondition is: the number of spots on the image is greater than a presetvalue, the first evaluation value of the image at a correspondingposition is not greater than a first threshold, and the secondevaluation value of the image at the corresponding position is maximalamong the second evaluation values of N images before and after theimage at the corresponding position; or the preset condition is: thenumber of spots on the image is less than the preset value, the firstevaluation value of the image at the corresponding position is notgreater than the first threshold, and the third evaluation value of theimage at the corresponding position is maximal among the thirdevaluation values of N images before and after the current image.

In some embodiments, the system for imaging comprises a spot detectionmodule configured for: using k1×k2 matrices to detect the spots on theimage, comprising determining that the matrix in which a central pixelvalue of the matrix is not less than any non-central pixel value of thematrix corresponds to a spot, wherein both k1 and k2 are odd numbersgreater than 1, and the k1×k2 matrix comprises k1×k2 pixels.

In some embodiments, the central pixel value of the matrix correspondingto a spot is greater than a first preset value, any non-central pixelvalue of the matrix is greater than a second preset value, and the firstpreset value and the second preset value are related to an average pixelvalue of the image.

In some embodiments, the first evaluation value is determined bycalculating the sizes of connected components corresponding to the spotsof the image, the size Area of the connected component corresponding toa spot of the image is equal to A×B with A representing the size of theconnected component in row centered on the center of the matrixcorresponding to the spot and B representing the size of the connectedcomponent in column centered on the center of the matrix correspondingto the spot, and a connected component is defined as pixels connectivitygreater than the average pixel value of the image.

In some embodiments, the second evaluation value and/or the thirdevaluation value is determined by calculating scores of the spots of theimage, and the score of the spot of one image is equal to((k1×k2−1)CV−EV)/((CV+EV)/(k1×k2)), wherein CV represents the centralpixel value of the matrix corresponding to the spot, and EV representsthe sum of the non-central pixel values of the matrix corresponding tothe spot.

In some embodiments, the focusing module 106 comprises a light source116 and an optical sensor 118, the light source 116 is configured foremitting light onto an object, and the optical sensor 118 is configuredfor receiving light reflected by the object.

In some embodiments, when the focusing module 106 receives the lightreflected by the object, the control device 101 is further configuredfor: moving the lens 104 toward the object at a third set step lengthless than the first set step length and greater than the second set steplength, calculating a first light intensity parameter according to thelight intensity of the light received by the focusing module 106, anddetermining whether the first light intensity parameter is greater thana first set light intensity threshold; and moving the lens 104 from thecurrent position to the second set position when the first lightintensity parameter is greater than the first set light intensitythreshold.

In some embodiments, the focusing module 106 comprises two opticalsensors 118, the two optical sensors 118 are configured for receivingthe light reflected by an object, and the first light intensityparameter is an average value of light intensities of light received bythe two optical sensors 118.

In some embodiments, as the lens 104 moves, the control device 101 isconfigured for: determining whether the current position of the lens 104is beyond a fourth set position; and when the current position of thelens 104 is beyond the fourth set position, stopping the movement of thelens 104.

In some embodiments, the control device 101 is configured for: emittinglight onto the object using the focusing module 106; moving the lens 104to a first set position; moving the lens 104 from the first set positiontoward the object at a first set step length and determining whether thefocusing module 106 receives the light reflected by the object; when thefocusing module 106 receives the light reflected by the object, movingthe lens 104 at a second set step length less than the first set steplength, acquiring an image of the object using the device for imaging102, and determining whether a sharpness value of the image acquired bythe device for imaging 102 reaches a set threshold; and when thesharpness value of the image reaches the set threshold, saving thecurrent position of the lens 104 as a saved position.

In some embodiments, the focusing module 106 comprises a light source116 and an optical sensor 118, the light source 116 is configured foremitting light onto an object, and the optical sensor 118 is configuredfor receiving light reflected by the object.

In some embodiments, when the focusing module 106 receives the lightreflected by the object, the control device 101 is configured for:moving the lens 104 toward the object at a third set step length lessthan the first set step length and greater than the second set steplength, calculating a first light intensity parameter according to thelight intensity of the light received by the focusing module 106, anddetermining whether the first light intensity parameter is greater thana first set light intensity threshold; and when the first lightintensity parameter is greater than the first set light intensitythreshold, moving the lens 104 at the second set step length, acquiringan image of the object using the device for imaging 102, and determiningwhether a sharpness value of the image acquired by the device forimaging 102 reaches a set threshold.

In some embodiments, the focusing module 106 comprises two opticalsensors 118, the two optical sensors 118 are configured for receivingthe light reflected by an object, and the first light intensityparameter is an average value of light intensities of light received bythe two optical sensors 118.

In some embodiments, when the focusing module 106 receives the lightreflected by the object, the control device 101 is configured for:moving the lens 104 toward the object at a third set step length lessthan the first set step length and greater than the second set steplength, calculating a first light intensity parameter according to thelight intensity of the light received by the focusing module 106, anddetermining whether the first light intensity parameter is greater thana first set light intensity threshold; and when the first lightintensity parameter is greater than the first set light intensitythreshold, moving the lens 104 toward the object at a fourth set steplength less than the third set step length and greater than the secondset step length, calculating a second light intensity parameteraccording to the light intensity of the light received by the focusingmodule 106, and determining whether the second light intensity parameteris less than a second set light intensity threshold; and when the secondlight intensity parameter is less than the second set light intensitythreshold, moving the lens 104 at the second set step length, acquiringan image of the object using the device for imaging 102, and determiningwhether a sharpness value of the image acquired by the device forimaging 102 reaches a set threshold.

In some embodiments, the focusing module 106 comprises two opticalsensors 118, the two optical sensors 118 are configured for receivingthe light reflected by the object, and the first light intensityparameter is an average value of the light intensities of the lightreceived by the two optical sensors 118; the light intensities of thelight received by the two optical sensors 118 have a first difference,and the second light intensity parameter is a difference between thefirst difference and a set compensated value.

In some embodiments, when the lens 104 is moved at the second set steplength, the control device 101 is configured for: determining whetherthe first sharpness value of the image corresponding to the currentposition of the lens 104 is greater than the second sharpness value ofthe image corresponding to the previous position of the lens 104; whenthe first sharpness value is greater than the second sharpness value anda sharpness difference between the first sharpness value and the secondsharpness value is greater than a set difference, moving the lens 104toward the object at the second set step length; when the firstsharpness value is greater than the second sharpness value and thesharpness difference between the first sharpness value and the secondsharpness value is less than the set difference, moving the lens 104toward the object at a fifth set step length less than the second setstep length such that the sharpness value of the image acquired by thedevice for imaging 102 reaches the set threshold; when the secondsharpness value is greater than the first sharpness value and thesharpness difference between the second sharpness value and the firstsharpness value is greater than the set difference, moving the lens 104away from the object at the second set step length; and when the secondsharpness value is greater than the first sharpness value and thesharpness difference between the second sharpness value and the firstsharpness value is less than the set difference, moving the lens 104away from the object at the fifth set step length such that thesharpness value of the image acquired by the device for imaging 102reaches the set threshold.

In some embodiments, as the lens 104 moves, the control device 101 isconfigured for: determining whether the current position of the lens 104is beyond a second set position; and when the current position of thelens 104 is beyond the second set position, stopping the movement of thelens 104 or performing focusing.

A computer-readable storage medium according to an embodiment of thepresent disclosure is configured for storing a program for execution bya computer, and the execution of the program comprises implementing thesteps of the method for imaging according to any of the aforementionedembodiments. The computer-readable storage medium may include: read-onlymemory, random access memory, magnetic disk, optical disk, or the like.

A computer program product according to an embodiment of the presentdisclosure comprises instructions, which enable a computer to implementthe steps of the method for imaging according to any of theaforementioned embodiments when executed by the computer.

In the description of this specification, the description of the terms“one embodiment”, “some embodiments”, “schematic embodiments”,“examples”, “specific examples”, “some examples” or the like, means thatthe particular features, structures, materials or characteristicscomprised in the embodiments or examples are included in at least oneembodiment or example of the present disclosure. In this specification,the schematic description of the aforementioned terms do not necessarilyrefer to the same embodiment or example. Moreover, the particularfeatures, structures, materials or characteristics described may becombined in any embodiment or example in any appropriate manner.

In addition, each functional unit in each embodiment of the presentdisclosure may be integrated in one processing module, or each unit mayphysically exist alone, or two or more than two units may be integratedin one module. The above-mentioned integrated module may be implementedin the form of hardware or in the form of software functional modules.The integrated module may also be stored in a computer-readable storagemedium if it is implemented in the form of a software functional moduleand is sold or used as standalone products.

Although the embodiments of the present disclosure have been shown anddescribed above, it is to be understood that the aforementionedembodiments are exemplary and are not to be construed as limiting thepresent disclosure, and that those of ordinary skill in the art may makechanges, modifications, replacements and variations to such embodiments,without departing from the scope of the present disclosure.

What is claimed is:
 1. A method of imaging, wherein the method uses asystem for imaging to image an object, the system for imaging comprisesa lens, and the object comprises a first object, a second object and athird object located at different positions on a first preset track, andthe object comprises a fourth object and a fifth object located atdifferent positions on a second preset track, the method comprising:allowing the lens and the first preset track to move relatively in afirst predetermined relationship to acquire a clear image of the thirdobject using the system for imaging without focusing, wherein the firstpredetermined relationship is determined by a focal plane position ofthe first object and a focal plane position of the second object; andallowing the lens and the second preset track to move relatively in asecond predetermined relationship to acquire a clear image of the fifthobject using the system for imaging without focusing, wherein the secondpredetermined relationship is determined by a focal plane position ofthe fourth object and the first predetermined relationship, and thesecond preset track is different from the first preset track.
 2. Themethod according to claim 1, wherein the third object is located betweenthe first object and the second object.
 3. The method according to claim1, wherein the determination of the first predetermined relationshipcomprises: focusing on the first object using the system for imaging todetermine first coordinates; focusing on the second object using thesystem for imaging to determine second coordinates; and establishing thefirst predetermined relationship according to the first coordinates andthe second coordinates, wherein the first coordinates represent thefocal plane position of the first object, and the second coordinatesrepresent the focal plane position of the second object.
 4. The methodaccording to claim 1, wherein the first preset track is a linear ornonlinear track; or the first predetermined relationship is a linearrelationship.
 5. The method according to claim 1, wherein the lens isfixed and comprises an optical axis, and any or both of the first presettrack and the second preset track can move in a direction perpendicularor parallel to the optical axis.
 6. The method according to claim 1,wherein the determination of the second predetermined relationshipcomprises: focusing on the fourth object using the system for imaging todetermine fourth coordinates; and establishing the second predeterminedrelationship according to the first predetermined relationship and thefourth coordinates, wherein the fourth coordinates represent the focalplane position of the fourth object.
 7. The method according to claim 1,comprising: allowing, after acquiring the clear image of the thirdobject, the lens and the first preset track or the second preset trackto move relatively to acquire the clear image of the fifth object usingthe system for imaging without focusing.
 8. The method according toclaim 6, wherein the system for imaging comprises a device for imagingand a carrier, the device for imaging comprises the lens and a focusingmodule, the lens comprises an optical axis and can move in the directionof the optical axis to perform the focusing, and the first preset trackand the second preset track are located on the carrier.
 9. The methodaccording to claim 8, wherein the focusing comprises: (a) emitting lightonto the object using the focusing module; (b) moving the lens to afirst set position; (c) moving the lens from the first set positiontoward the object at a first set step length and determining whether thefocusing module receives the light reflected by the object; (d) when thefocusing module receives the light reflected by the object, moving thelens from current position to a second set position, wherein the secondset position is located within a first range, and the first range is arange comprising the current position and allowing the lens to move inthe direction of the optical axis; (e) moving the lens from the secondset position at a second set step length and acquiring an image of theobject at a position of each step using the device for imaging, whereinthe second set step length is less than the first set step length; and(f) evaluating the images of the object to obtain an image evaluationresult and focusing according to the image evaluation result.
 10. Themethod according to claim 9, wherein, with the current position as areference, the first range comprises a first interval and a secondinterval opposite to each other, the second interval is defined closerto the object, and step (e) comprises: (i) when the second set positionis located within the second interval, moving the lens from the secondset position in a direction away from the object and acquiring an imageof the object at a position of each step using the device for imaging;or (ii) when the second set position is located within the firstinterval, moving the lens from the second set position in a directiontoward the object and acquiring an image of the object at a position ofeach step using the device for imaging.
 11. The method according toclaim 10, wherein step (f) comprises: comparing the image evaluationresult with a preset condition, and if the image evaluation result meetsthe preset condition, saving the position of the lens corresponding tothe image; and if the image evaluation result does not meet the presetcondition, moving the lens to a third set position, wherein the thirdset position is located within the other interval of the first rangedifferent from the interval where the second set position is located.12. The method according to claim 11, wherein the image evaluationresult comprises a first evaluation value and a second evaluation value,the second set step length comprises a rough step length and a fine steplength, and step (f) comprises: moving the lens at the rough step lengthuntil the first evaluation value of the image at a correspondingposition is not greater than a first threshold; moving the lens at thefine step length until the second evaluation value of the image at acorresponding position is maximal; and saving the position of the lenscorresponding to the image when the second evaluation value is themaximum value.
 13. The method according to claim 11, wherein the imageevaluation result comprises a first evaluation value, a secondevaluation value and a third evaluation value, and the image comprises aplurality of pixels; the preset condition is: the number of spots on theimage is greater than a preset value, the first evaluation value of theimage at a corresponding position is not greater than a first threshold,and the second evaluation value of the image at the correspondingposition is maximal among the second evaluation values of N imagesbefore and after the image at the corresponding position; or the presetcondition is: the number of spots on the image is less than the presetvalue, the first evaluation value of the image at the correspondingposition is not greater than the first threshold, and the thirdevaluation value of the image at the corresponding position is maximalamong the third evaluation values of N images before and after thecurrent image.
 14. The method according to claim 13, wherein thefollowing method is used to detect the spots on the image: using k1×k2matrices to detect the spots on the image, comprising determining thatthe matrix in which a central pixel value of the matrix is not less thanany non-central pixel value of the matrix corresponds to a spot, whereinboth k1 and k2 are odd numbers greater than 1, and the k1×k2 matrixcomprises k1×k2 pixels.
 15. The method according to claim 14, whereinthe central pixel value of the matrix corresponding to a spot is greaterthan a first preset value, any non-central pixel value of the matrix isgreater than a second preset value, and the first preset value and thesecond preset value are related to an average pixel value of the image.16. The method according to claim 15, wherein the first evaluation valueis determined by calculating the sizes of connected componentscorresponding to the spots of the image, the size Area of the connectedcomponent corresponding to a spot of the image is equal to A×B with Arepresenting the size of the connected component in row centered on thecenter of the matrix corresponding to the spot and B representing thesize of the connected component in column centered on the center of thematrix corresponding to the spot, and a connected component is definedas pixels connectivity greater than the average pixel value of theimage.
 17. The method according to claim 14, wherein the secondevaluation value or the third evaluation value is determined bycalculating scores of the spots of the image, and the score of a spot ofthe image is equal to ((k1×k2−1)CV−EV)/((CV+EV)/(k1×k2)), CVrepresenting the central pixel value of the matrix corresponding to thespot and EV representing the sum of the non-central pixel values of thematrix corresponding to the spot.
 18. The method according to claim 8,wherein, when the focusing module receives the light reflected by theobject, the focusing further comprises the following steps: moving thelens toward the object at a third set step length less than the firstset step length and greater than the second set step length; calculatinga first light intensity parameter according to the light intensity ofthe light received by the focusing module; determining whether the firstlight intensity parameter is greater than a first set light intensitythreshold; and moving the lens from the current position to the secondset position when the first light intensity parameter is greater thanthe first set light intensity threshold.
 19. A sequencing device forsequencing nucleic acid molecules, wherein the sequencing devicecomprises a lens and a control device, the nucleic acid moleculescomprise first nucleic acid molecules, second nucleic acid molecules andthird nucleic acid molecules located at different positions on a firstpreset track, and the nucleic acid molecules further comprise fourthnucleic acid molecules and fifth nucleic acid molecules located atdifferent positions on a second preset track, and wherein the controldevice is configured to implement actions comprising: allowing the lensand the first preset track to move relatively in a first predeterminedrelationship to acquire a clear image of the third nucleic acidmolecules using the sequencing device without focusing, wherein thefirst predetermined relationship is determined by a focal plane positionof the first nucleic acid molecules and a focal plane position of thesecond nucleic acid molecules; and allowing the lens and the secondpreset track to move relatively in a second predetermined relationshipto acquire a clear image of the fifth nucleic acid molecules using thesequencing device without focusing, wherein the second predeterminedrelationship is determined by a focal plane position of the fourthnucleic acid molecules and the first predetermined relationship, and thesecond preset track is different from the first preset track.